Deuterated compounds and uses thereof

ABSTRACT

An anthraquinone compound of formula I (such as the compounds of formulae II to X) and processes for making the same are provided. Pharmaceutical compositions for use in the treatment of cancer, optionally in combination with an agent capable of reducing the level of oxygenation of a tumor, are also provided. Additionally, an option for combination with chemotherapeutic and radiotherapeutic modalities to enhance overall tumor cell kill is provided. Methods for the detection of cellular hypoxia, both in vivo and in vitro, are additionally provided.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to Great Britain PatentApplication No. 1214169.3, filed Aug. 8, 2012, incorporated herein inits entirety.

TECHNICAL FIELD

The present invention relates to novel anthraquinone compounds and usesof the same, for example in the treatment of cancer.

BACKGROUND

The therapeutic advantage of an anticancer drug depends primarily on theextent to which the agent shows selective activity for tumour cells andthe limiting toxicity towards non-target tissues. Frequently the poorquality of the vasculature within the growing tumour mass compromisesthe delivery of drugs, nutrients and oxygen. It is recognised thattumours can have significantly lower median oxygen levels (approximately1% oxygen; pO2 7.5 mmHg) compared to normal tissues (˜5.5% oxygen; 42mmHg) (summarised from data presented by Brown and Wilson, 2004). Inaddition, oxygenation levels can vary throughout the tumour due tointermittent opening and closing of tumour blood vessels; poorvascularisation, especially in the tumour core, contributes to oxygenlevels often being below 0.1% oxygen (1 mm Hg). Tumour cellsexperiencing varying degrees of hypoxia, relative to normally perfusedtissues, can compromise treatment effectiveness and contribute to themalignancy. Hypoxia-selective agents (e.g. bioreductive drugs) compriseone class of agents that can be used to target tumour cells in very lowoxygen environments by virtue of a selective activation to a cytotoxicform under reduced oxygenation, addressing the problems of non-targettissue toxicity, hypoxic cell drug resistance and cancer progression.

Poor oxygenation results in a relative state of hypoxia when comparedwith normoxic conditions in which oxygenation has not been compromised.Poor oxygenation within tumours can modify the responses to treatmentmodalities and contribute to cancer progression. Cells in such hypoxicareas are particularly resistant to treatment with many of theconventionally used anticancer drugs; this is attributed to poor drugdelivery and/or lack of intrinsic tumour cell sensitivity of viable butquiescent cells. Radiotherapy is also less effective at very low oxygenlevels since the cytotoxicity of ionising radiation is enhanced by thepresence of oxygen (Radiobiology For The Radiologist, Hall E J, GiacciaA J, Lippincott Williams & Wilkins, (2005)). Recent evidence shows thattumour cells can adapt to low oxygen conditions and change thepharmacodynamic responses to anticancer agents through the induction ofactive cellular protective mechanisms (Vaupel and Mayer 2007, CancerMetastasis Rev 26(2): 225-239). Additionally, it is recognized thattumour cells that survive hypoxic stress often show a more malignantmetastatic phenotype (Vaupel P, Metabolic microenvironment of tumorcells: a key factor in malignant progression, Exp Oncol 2010; 32,125-127); this has significant consequences for the patient. Followingtreatment with modalities that target predominantly thebetter-oxygenated cells, the stress-resistant hypoxic cells oftenrepopulate the tumour with cells that have an enhanced potential tospread to distant tissues. The development of more malignant metastatictumours is often the precursor to a more significant disease-relatedmorbidity and the death of the patient.

An attractive approach is the use of a hypoxia activated prodrug that isnon-toxic towards adequately oxygenated cells found in systemic tissues,but becomes activated or converted to a cytotoxic form under reducedoxygenation conditions. N-oxide derivatives of cytotoxicalkylaminoanthraquinones provide anthraquinone pro-drugs that showalmost no cytotoxicity. Importantly these prodrugs are capable of beingconverted in vivo under the anaerobic/hypoxic conditions found withinneoplastic tissue. Specificity for the tumour is ensured since systemictissues, except for tumours, almost never experience oxygen levels lowenough to facilitate the production of the cytotoxic drug.

The anthraquinone N-oxide AQ4N (CAS#136470-65-0) is a prodrug that isselectively bioreduced to AQ4, a potent DNA topoisomerase II inhibitor,in hypoxic tumour cells. Previous publications have taught thefundamental properties and in-vitro/in-vivo characteristics of theprodrug AQ4N (for example, see U.S. Pat. No. 5,132,327).

The invention seeks to address the need for improved cancer treatmentsby providing novel anthraquinone compounds with a combination ofpreferable pharmacological and hypoxia-sensing properties.

SUMMARY

The first aspect of the invention provides a compound of Formula I

wherein X₁, X₂, X₃ and X₄ are each independently selected from the groupconsisting of hydrogen, hydroxy, halogeno, amino, C₁₋₄ alkoxy, C₂₋₈alkanoyloxy, —NH-A-NHR, —NH-A-NR′R″ and —NH-A-N(O)R′R″wherein A is an alkylene group with a chain length of at least twocarbon atoms (between NH and NHR or N(O)R′R″),wherein R, R′ and R″ are each independently selected from C₁₋₄ alkylgroups and C₂₋₄ hydroxyalkyl and C₂₋₄ dihydroxyalkyl groups in which thecarbon atom attached to the nitrogen atom does not carry a hydroxy groupand no carbon atom is substituted by two hydroxy groups, or wherein Rand R″ together are a C₂₋₆ alkylene group which with the nitrogen atomto which R′ and R″ are attached forms a heterocyclic group having 3 to 7atoms in the ring,wherein at least one of X₁, X₂, X₃ and X₄ is selected from the groupconsisting of deuterated forms of —NH-A-NHR, —NH-A-NR′R″ and—NH-A-N(O)R′R″.

Thus, the invention provides novel deuterated anthraquinone compounds.

By “deuterated” we include that the compound comprises at least one atomof deuterium or heavy hydrogen (i.e. D or ²H). It will be appreciated bypersons skilled in the art that the compound may be partially (i.e.selectively) or fully deuterated (i.e. containing hydrogen present onlyin the form of deuterium).

By “selectively”, in this context, we mean that some but not allconventional ¹H hydrogen atoms are replaced with deuterium. For example,one or more of substituent groups X₁, X₂, X₃ and X₄ may be deuteratedwhile the central anthraquinone ring may be free of deuterium.

In one embodiment, the compound of the invention is selectivelydeuterated within one or more of substituent groups —NH-A-NHR,—NH-A-NR′R″ and/or —NH-A-N(O)R′R″ at positions X₁, X₂, X₃ and/or X₄.Within each such substituent group, it will be appreciated that A, R, R′and R″ may be fully deuterated (i.e. thus containing no ¹H) or may bepartially deuterated.

In a preferred embodiment, the compound is deuterated only within one ormore of the terminal groups R, R′ and R″. For example, R, R′ and/or R″may represent:

-   -   CD³;    -   CH₂CD³;    -   CD₂CD³;    -   CD₂CH₂CD³; and    -   CD₂CD₂ CD₂CD³.

The term “C₁₋₄ alkyl” is intended to include linear or branched alkylgroups comprising between one and four carbons. Preferred alkyl groupswhich R, R′ and/or R″ may independently represent include C₁ and C₂alkyl.

The term “lower alkylene” is to be construed accordingly.

The terms “C₂₋₄ hydroxyalkyl” and “C₂₋₄ dihydroxyalkyl” are intended toinclude linear or branched alkyl groups comprising between two and fourcarbons, to which are attached one or two hydroxy groups, respectively.For example, R, R′ and/or R″ may independently represent:

-   -   CH₂CH₂OH    -   CH₂CH(OH)CH₃    -   CH₂CH₂CH(OH)CH₂OH

The term “C₁₋₄ alkoxy” is intended to include linear or branched C₁₋₄alkyl groups bound to the core anthraquinone (anthracene-9,10-dione)ring via oxygen. For example, R, R′ and/or R″ may independentlyrepresent:

-   -   OCH₃    -   OCH₂CH₃    -   OCH₂CH₂CH₃    -   OCH₂CH₂CH₂CH₃

The term “C₂₋₈ alkanoyloxy” is intended to include linear or branchedC₂₋₈ alkanoyl groups bound to the core anthraquinone(anthracene-9,10-dione) ring via oxygen. For example, R, R′ and/or R″may independently represent:

-   -   O(O)CCH₃    -   O(O)CCH₂CH₃    -   O(O)CCH₂CH₂CH₃    -   O(O)CCH₂CH₂CH₂CH₃    -   O(O)CCH₂CH₂CH(CH₃)CH₃

The term “hydroxy” is intended to represent —OH.

The term “halogeno” is intended to represent any halogen group, such as—Br, —Cl and —F.

The term “amino” is intended to include primary amine groups, such as—NH₂.

It will be appreciated by persons skilled in the art that theanthraquinone ring of the compounds may be substituted by X₁, X₂, X₃ andX₄ at any of ring positions 1, 2, 3, 4, 5, 6, 7 or 8:

In one embodiment of the first aspect of the invention, the compound issubstituted at ring positions 1, 4, 5 and 8, in accordance with FormulaII:

In one embodiment, X₁, X₂, X₃ and X₄ are each separately selected fromthe group consisting of hydrogen, hydroxy, —NH-A-NHR, —NH-A-NR′R″,—NH-A-N(O)R′R″ and deuterated forms thereof.

In one embodiment, X₁, X₂, X₃ and X₄ are each separately selected fromthe group consisting of hydroxy, —NH-A-NR′R″, —NH-A-N(O)R′R″ anddeuterated forms thereof.

In one embodiment, X₁ and X₂ are both hydroxy and X₃ and X₄ are both—NH-A-N(O)R′R″ or deuterated forms thereof.

In one embodiment, X₁ and X₂ are both hydroxy and X₃ and X₄ are bothNH-A-NR′R″ or deuterated forms thereof.

In one embodiment, A is unbranched. For example, A may be ethylene.

In one embodiment, R, R′ and R″ are each independently selected from thegroup consisting of —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH₂CH₂OH, —CH₂CH₂CH₂OH,—CH(CH₃)CH₂OH, —CH₂CHOHCH₂OH and deuterated forms thereof.

In one embodiment, one or two of X₁, X₂, X₃ and X₄ are independentlyselected from the group consisting of —NH—(CH₂)₂—N(O)(CH₃)₂,—NH—(CH₂)₂—N(O)(CH₃)C₂H₅, —NH—(CH₂)₂—N(O)(C₂H₅)₂, —NH—(CH₂)₂—N(O)(CH₂CH₂OH)₂, —NH—(CH₂)₂—N(O)(CH₂CH₂CH₂OH)₂, —NH—(CH₂)₂—N(O)CH(CH₃)OH,—NH—(CH₂)₂—N(O)(CH₂CHOHCH₂OH)₂ and deuterated forms thereof.

In one embodiment, one or two of X₁, X₂, X₃ and X₄ are independentlyselected from the group consisting of —NH—(CH₂)₂—N(CH₃)₂,—NH—(CH₂)₂—N(CH₃)C₂H₅, —NH—(CH₂)₂—N(C₂H₅)₂, —NH—(CH₂)₂—N(CH₂CH₂OH)₂,—NH—(CH₂)₂—N(CH₂CH₂CH₂OH)₂, —NH—(CH₂)₂—NCH(CH₃)OH,—NH—(CH₂)₂—N(CH₂CHOHCH₂OH)₂ and deuterated forms thereof.

In one embodiment, the compound of the invention comprises one group—NH-A-N(O)R′R″ and one group —NH-A-NHR, the —NH-A-NHR group beingselected from —NH—(CH₂)₂—NHCH₃, —NH—(CH₂)₂—NHC₂H₅,—NH—(CH₂)₂—NHCH₂CH₂OH, —NH—(CH₂)₂—NHCH₂CH₂CH₂OH,—NH—(CH₂)₂—NHCH(CH₃)CH₂OH, —NH—(CH₂)₂—NHCH₂CHOHCH₂OH and deuteratedforms thereof.

In one embodiment, the compound of the invention comprises one group—NH-A-NR′R″ and one group —NH-A-NHR, the —NH-A-NHR group being selectedfrom —NH—(CH₂)₂—NHCH₃, —NH—(CH₂)₂—NHC₂H₅, —NH—(CH₂)₂—NHCH₂CH₂OH,—NH—(CH₂)₂—NHCH₂CH₂CH₂OH, —NH—(CH₂)₂—NHCH(CH₃)CH₂OH,—NH—(CH₂)₂—NHCH₂CHOHCH₂OH and deuterated forms thereof.

In preferred, but non-limiting, compounds of the invention:

(a) X₁=—NH-A-N(O)R′R″, X₂=—H and X₃=X₄=—OH;

(b) X₁=—NH-A-N(O)R′R″, X₂=—OH, X₃=—OH and X₄=—H;

(c) X₁=—NH-A-N(O)R′R″ and X₂=X₃=X₄=—OH;

(d) X₁=X₄=—NH-A-N(O)R′R″ and X₂=X₃=—OH;

(e) X₁=X₂=—NH-A-N(O)R′R″ and X₃=X₄=—OH;

(f) X₁=X₃=—NH-A-N(O)R′R″ and X₂=X₄=—OH;

(g) X₁=—NH-A-NR′R″, X₂=—H and X₃=X₄=—OH;

(h) X₁=—NH-A-NR′R″, X₂=—OH at position 4, X₃=—OH and X₄=—H;

(i) X₁=—NH-A-NR′R″ and X₂=X₃=X₄=—OH;

(j) X₁=X₄=—NH-A-NR′R″ and X₂=X₃=—OH;

(k) X₁=X₂=—NH-A-NR′R″ and X₃=X₄=—OH;

(l) X₁=X₃=—NH-A-NR′R″ and X₂=X₄=—OH;

and deuterated forms thereof.

In further preferred, but non-limiting, compounds of the invention:

(a) X₁=—NH-A-N(O)R′R″, X₂=—NH-A-NHR, and X₃=X₄=—OH;

(b) X₁=—NH-A-N(O)R′R″, X₂=—OH, X₃=—NH-A-NHR and X₄=—OH;

(c) X₁=—NH-A-N(O)R′R″, X₂=X₃=—OH and X₄=—NH-A-NHR;

(d) X₁=—NH-A-NR′R″, X₂=—NH-A-NHR, and X₃=X₄=—OH;

(e) X₁=—NH-A-NR′R″, X₂=—OH, X₃=—NH-A-NHR and X₄=—OH;

(f) X₁=—NH-A-NR′R″, X₂=X₃=—OH and X₄=—NH-A-NHR;

and deuterated forms thereof.

In further preferred, but non-limiting, compounds of the invention:

(a) X₁=X₂=—NH-A-N(O)R′R″ and X₃=X₄=—OH;

(b) X₁=X₃=—NH-A-N(O)R′R″ and X₂=X₄=—OH;

(c) X₁=X₂=—NH-A-NR′R″ and X₃=X₄=—OH; and

(d) X₁=X₃=—NH-A-NR′R″ and X₂=X₄=—OH

wherein

both —NH-A-N(O)R′R″ are —NH—(CH₂)₂N(O)(CH₃)₂ or—NH—(CH₂)₂N(O)(CH₂CH₂OH)₂, or deuterated forms thereof and

both NH-A-NR′R″ are —NH—(CH₂)₂N(CH₃)₂ or —NH—(CH₂)₂N(CH₂CH₂OH)₂, ordeuterated forms thereof.

In further preferred, but non-limiting, compounds of the invention:

(a) X₁=—NH-A-N(O)R′R″, X₂=—NH-A-NHR and X₃=X₄=—OH;

(b) X₁=—NH-A-N(O)R′R″, X₂=—OH, X₃=—NH-A-NHR and X₄=—OH;

(c) X₁=—NH-A-NR′R″, X₂=—NH-A-NHR and X₃=X₄=—OH; and

(d) X₁=—NH-A-NR′R″, X₂=—OH, X₃=—NH-A-NHR and X₄=—OH,

wherein

—NH-A-N(O)R′R″ is —NH—(CH₂)₂N(O)(CH₃)₂ or —NH—(CH₂)₂N(O)(CH₂CH₂OH)₂ or adeuterated form thereof

—NH-A-NHR is NH—(CH₂)₂NHCH₃ or NH(CH₂)₂NHCH₂CH₂OH or a deuterated formthereof

and NH-A-NR′R″ is —NH—(CH₂)₂N(CH₃)₂ or —NH—(CH₂)₂N(CH₂CH₂OH)₂ or adeuterated form thereof.

The present invention relates to novel anthraquinone compounds and usesof the same, for example in the treatment of cancer.

The present invention relates to novel anthraquinone compounds and usesof the same, for example in the treatment of cancer.

In one embodiment, the compound is of Formula III or IV:

wherein Y are each independently selected from the group consisting ofhydrogen, hydroxy, halogeno, amino, C₁₋₄ alkoxy and C₂₋₈ alkanoxy, or aprodrug thereof.

By “prodrug”, in this context, is included compounds which may readilybe converted in vivo to a compound of Formula III or IV. In oneembodiment, the conversion is triggered by the prodrug entering anhypoxic environment, such as a solid tumour.

Examples of suitable prodrugs include N-oxide derivatives of thecompounds of Formula III or IV.

Thus, in one embodiment, the prodrug is a compound of Formula V or VI:

wherein Y are each independently selected from the group consisting ofhydrogen, hydroxy, halogeno, amino, C₁₋₄ alkoxy and C₂₋₈ alkanoxy.

In one preferred embodiment, the compound is of Formula VII or VIII:

or a prodrug thereof.

The present invention relates to novel anthraquinone compounds and usesof the same, for example in the treatment of cancer.

The present invention relates to novel anthraquinone compounds and usesof the same, for example in the treatment of cancer.

In a further preferred embodiment, the compound is prodrug of Formula IXor X:

In the compounds of Formulae III to X, it will be appreciated by personsskilled in the art that one or more of the deuterium atoms in one ormore of the methyl groups attached to the nitrogen of the terminal aminogroups may be replaced by conventional hydrogen (i.e. ¹H), provided thatthe compound comprises at least one deuterium atom. For example, one,two, three or four of the methyl groups may be —CH₃, —CH₂D or —CHD₂. Inone embodiment, the methyl groups in the compound are either —CH₃ or—CD₃.

It will be further appreciated by skilled persons that certain compoundsof formulae I to X above may be counterbalanced by counter-anions.Exemplary counter-anions include, but are not limited to, halides (e.g.fluoride, chloride and bromide), sulfates (e.g. decylsulfate), nitrates,perchlorates, sulfonates (e.g. methane sulfonate) and trifluoroacetate.Other suitable counter-anions will be well known to persons skilled inthe art. Thus, pharmaceutically, and/or veterinarily, acceptablederivatives of the compounds of formulae I to X, such as salts andsolvates, are also included within the scope of the invention. Saltswhich may be mentioned include: acid addition salts, for example, saltsformed with inorganic acids such as hydrochloric, hydrobromic, sulfuricand phosphoric acid, with carboxylic acids or with organo-sulfonicacids; base addition salts; metal salts formed with bases, for example,the sodium and potassium salts.

In one embodiment, the compound is in the form of a halide salt, forexample a chloride salt.

It will be further appreciated by skilled persons that certain compoundsof formulae I to X may exhibit tautomerism. All tautomeric forms andmixtures thereof are included within the scope of the invention.

Compounds of formulae I to X may also contain one or more asymmetriccarbon atoms and may therefore exhibit optical and/ordiastereoisomerism. Diastereoisomers may be separated using conventionaltechniques, e.g. chromatography or fractional crystallisation. Thevarious stereoisomers may be isolated by separation of a racemic orother mixture of the compounds using conventional, e.g. fractionalcrystallisation or HPLC, techniques. Alternatively, the desired opticalisomers may be made by reaction of the appropriate optically activestarting materials under conditions which will not cause racemisation orepimerisation, or by derivatisation, for example with a homochiral acidfollowed by separation of the diastereomeric esters by conventionalmeans (e.g. HPLC, chromatography over silica). All stereoisomers areincluded within the scope of the invention.

Various routes are available for the synthesis of the compounds of theinvention. One very convenient procedure for the preparation ofcompounds having a group —NH-A-NR′R″ at the 1 and 4 positions uses theappropriately substituted2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione which is condensedwith the appropriate amine R″R′N—A—NH₂, the 1,4 positions beingactivated in the leuco compound for reaction with the amine. Such acondensation may conveniently be effected at a temperature in a range ofabout 25° C. or 35° C. to 50° C. or 60° C. for one or more hours using asolvent such as methanol, ethanol, water, dimethylformamide,2-methoxyethanol, acetonitrile, nitrobenzene,N,N,N′N′-tetra-methylenediamine or mixtures thereof. In some instances ahigher temperature and shorter reaction time may be appropriate, forexample with the compounds containing cyclic groups NR′R″. The leucoderivative is then oxidized to the fully aromatic anthracene-9,10-dione,conveniently using air oxidation or oxidation with hydrogen peroxide,chloranil, sodium perborate or manganese dioxide.

Although leuco compounds are primarily of interest for the preparationof compounds substituted by two —NH-A-NHR′R″ groups, it is possible touse them to prepare compounds containing more than two such groups.Thus, by using2,3-dihydro(leuco)-1,4,5,8-tetrahydroxyanthracene-9,10-dione and a largeexcess of an amine —NH-A-NHR′R″ an 8-hydroxyanthracene-9,10-dione havingthree groups —NH-A-NHR′R″ at the 1, 4 and 5 positions may be prepared.

The leuco derivatives themselves are obtainable by heat treatment of thecorresponding fully aromatic 1,4-dihydroxyanthracene-9,10-dione,conveniently by heating at above 90° C. for 1 hour or more in a streamof nitrogen and, if necessary, in the presence of a suitable reducingagent such as sodium dithionite or zinc dust. Variousanthracene-9,10-diones, particularly hydroxyanthracene-9,10-diones, arecommercially available and various syntheses for such compounds are alsoreported in the literature. One suitable procedure for their preparationinvolves the reaction of an appropriately substituted phthalic anhydridewith hydroquinone in the presence of aluminium chloride and sodiumhydroxide at 180° C. for one hour or more. Anthracene-9,10-dionescontaining one form of substituent group can be modified to provideother forms of substituent group so that, for example, a dionecontaining an amino group can be treated with sodiumhydroxide/dithionite to yield the corresponding hydroxy substitutedcompound.

Other suitable procedures for the preparation of intermediates foroxidation to the N-oxide compounds of the invention include the reactionof the appropriate chloro or fluoro substituted anthracene-9,10-dionewith the appropriate amine R″R′N—A—NH₂, for example by heating with aexcess of the amine at its reflux temperature for one or more hours.Certain of these chloro- and fluoro anthracene-9,10-diones are known andvarious syntheses for such compounds are also reported in theliterature. Thus, for example, a KF—NaF-mediated conversion of3,6-dichlorophthalic anhydride to 3,6-difluorophthalic anhydride as aprecursor to making 1,4-difluoro-4,8-dihydroxyanthracene-9,10-dione (seeLee & Denny, 1999, J. Chem. Soc., Perkin Trans. 1:2755-2758.Additionally, for example,1,5-dichloro-4,8-dihydroxyanthracene-9,10-dione may be prepared byselective chlorination of 1,4,5,8-tetrahydroxyanthracene-9,10-dioneusing a stoichiometric amount of sulphuryl chloride and controlledtemperature. This precursor may then be used to prepare an intermediatehaving groups —NH-A-NR′R″ at the 1 and 5 positions and hydroxy groups atthe 4 and 8 positions, the hydroxy groups conveniently being protectedduring the reaction with the amine R″R′N—A—NH₂. A similar approach issuitable for the preparation of other chlorohydroxyanthracene-9,10-dioneintermediates.

Where the compound of the invention contains one or more groups—NH-A-NHR in addition to the one or more groups —N-A-NR′R″ the compoundmay conveniently be produced by reacting a suitable precursor asdiscussed above with a mixture of amines RN—A—NH.sub.2 andR″R′N—A—NH.sub.2, the resultant mixture of products then beingseparated, for example by chromatography. Thus, for example,2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione on reaction with amixture of 2-(2-hydroxyethylamino)ethylamine and2-(diethylamino)ethylamine will yield a mixture of1,4-bis{[2-(diethylamino)-ethyl]amino}anthracene-9,10-dione,1,4-bis{[2-(2-hydroxyethyl-amino)-ethyl]amino}-anthracene-9,10-dione and1-(2-(diethylamino)ethyl]amino)-4-{[2-(2-hydroxyethylamino)-ethyl]amino}anthracene-9,10-dionefrom which the last mentioned compound may be separated, for example bychromatography. On oxidation, only the tertiary nitrogen atom of the[2-(diethylamino)ethyl)] amino group will be converted to N-oxide form.

Where one or more substituent groups is present it may be appropriate,depending on the route of synthesis, to have these present throughout intheir final form or to generate the desired groups at a later stage inthe synthesis. Ether and ester groups X may of course readily beprepared by modification of hydroxy groups according to knownprocedures, precursors containing a hydroxy group X more often beingdescribed in the literature than those containing a corresponding etheror ester substituent.

It will be appreciated, however, that various alternative methods forthe preparation of the compounds and intermediates therefor may be usedas will be apparent in particular from the literature relating to suchintermediates. Further details of the preparation of intermediates forthe preparation of the compounds of the present invention are to befound in U.S. Pat. No. 4,197,249 and GB 2,004,293B (the disclosures ofwhich are incorporated herein by reference).

Thus, a second aspect of the invention provides a process for making acompound according to the first aspect of the invention comprisingreacting an anthracene-9,10-dione with a deuterated alkylenediamineunder conditions suitable for the production of analkylaminoalkylaminoanthraquinone.

Optionally, the process further comprises the step of reacting thealkylaminoalkylaminoanthraquinone with a monoperoxyphthalate to underconditions suitable for the production of an N-oxide derivative of thealkylamino-alkylaminoanthraquinone.

In one embodiment, the process comprises reacting1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, 281-005 withdeuterated—N,N-dimethylethylene-diamine under conditions suitable forthe production of1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione.

In a further embodiment, the process comprises the step of reacting the1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dionewith magnesium monoperoxyphthalate under conditions suitable for theproduction of1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione.

A third aspect of the invention provides a pharmaceutical compositioncomprising a compound according to the first aspect of the inventiontogether with pharmaceutically acceptable buffer, diluent, carrier,adjuvant or excipient.

By “pharmaceutically acceptable” we include a non-toxic material thatdoes not decrease the therapeutic effectiveness of the compound of theinvention. Such pharmaceutically acceptable buffers, carriers orexcipients are well-known in the art (see Remington's PharmaceuticalSciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company(1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe,Ed., Pharmaceutical Press (2000), the disclosures of which areincorporated herein by reference).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the agent in the pharmaceuticalpreparation. The diluent may be one or more of saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the compound of theinvention. The adjuvant may be one or more of zinc, copper or silversalts with different anions, for example, but not limited to fluoride,chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate,carbonate, lactate, glycolate, citrate, borate, tartrate, and acetatesof different acyl composition. The adjuvant may also be cationicpolymers such as cationic cellulose ethers, cationic cellulose esters,deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationicsynthetic polymers such as poly(vinyl imidazole), and cationicpolypeptides such as polyhistidine, polylysine, polyarginine, andpeptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, glucose, sucrose,mannitol, and cyclodextrines, which are added to the composition, e.g.,for facilitating lyophilisation. Examples of polymers are starch,cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose,alginates, carageenans, hyaluronic acid and derivatives thereof,polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The compounds of the invention may be formulated into any type ofpharmaceutical composition known in the art to be suitable for thedelivery thereof.

In one preferred embodiment, the pharmaceutical compositions areadministered parenterally, for example, intravenously,intracerebroventricularly, intraarticularly, intraarterially,intraperitoneally, intrathecally, intraventricularly, intrasternally,intracranially, intramuscularly or subcutaneously, or they may beadministered by infusion techniques. The pharmaceutical compositions mayalso administered intra-tumourally and/or peri-tumourally.

Such pharmaceutical compositions are conveniently used in the form of asterile aqueous solution which may contain other substances, forexample, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well known to those skilled in theart.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

In a further embodiment, the pharmaceutical compositions of theinvention may be in the form of a liposome, in which the agent iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids, which exist in aggregated formsas micelles, insoluble monolayers and liquid crystals. Suitable lipidsfor liposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Suitable lipids also include the lipids abovemodified by poly(ethylene glycol) in the polar headgroup for prolongingbloodstream circulation time. Preparation of such liposomal formulationsis can be found in for example U.S. Pat. No. 4,235,871, the disclosuresof which are incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the formof biodegradable microspheres. Aliphatic polyesters, such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA)or poly(caprolactone) (PCL), and polyanhydrides have been widely used asbiodegradable polymers in the production of microspheres. Preparationsof such microspheres can be found in U.S. Pat. No. 5,851,451 and in EP 0213 303, the disclosures of which are incorporated herein by reference.

In a further embodiment, the pharmaceutical compositions of theinvention are provided in the form of polymer gels, where polymers suchas starch, cellulose ethers, cellulose carboxymethylcellulose,hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethylcellulose, alginates, carageenans, hyaluronic acid and derivativesthereof, polyacrylic acid, polyvinyl imidazole, polysulphonate,polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropyleneoxide copolymers, polyvinylalcohol/polyvinylacetate of different degreeof hydrolysis, and polyvinylpyrrolidone are used for thickening of thesolution containing the agent. The polymers may also comprise gelatin orcollagen.

Alternatively, the compounds may simply be dissolved in saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil),tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of theinvention may include ions and a defined pH for potentiation of actionof the active agent. Additionally, the compositions may be subjected toconventional pharmaceutical operations such as sterilisation and/or maycontain conventional adjuvants such as preservatives, stabilisers,wetting agents, emulsifiers, buffers, fillers, etc.

The pharmaceutical compositions according to the invention may beadministered via any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous,subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar,buccal, oral, parenteral, vaginal and rectal. Also administration fromimplants is possible.

Alternatively, the pharmaceutical compositions may be administeredintranasally or by inhalation (for example, in the form of an aerosolspray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas). In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activepolypeptide, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. A ‘therapeutically effective amount’,or ‘effective amount’, or ‘therapeutically effective’, as used herein,refers to that amount which provides a therapeutic effect for a givencondition and administration regimen. This is a predetermined quantityof active material calculated to produce a desired therapeutic effect inassociation with the required additive and diluent, i.e. a carrier oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent, a clinicallysignificant deficit in the activity, function and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity. Suitable dosage amounts maycontain a predetermined quantity of active composition calculated toproduce the desired therapeutic effect in association with the requireddiluent. In the methods and use for manufacture of compositions of theinvention, a therapeutically effective amount of the active component isprovided. A therapeutically effective amount can be determined by theordinary skilled medical or veterinary worker based on patientcharacteristics, such as age, weight, sex, condition, complications,other diseases, etc., as is well known in the art. The administration ofthe pharmaceutically effective dose can be carried out both by singleadministration in the form of an individual dose unit or else severalsmaller dose units and also by multiple administrations of subdivideddoses at specific intervals. Alternatively, the dose may be provided asa continuous infusion over a prolonged period.

It will be appreciated that the compositions of the invention may beformulated in unit dosage form, i.e. in the form of discrete portionscontaining a unit dose or a multiple or sub-unit of a unit dose.

Whilst the dosage of the compound used will vary according to theactivity of the particular compound and the condition being treated, itmay be stated by way of guidance that a dosage selected in the rangefrom 0.1 to 20 mg/kg per body weight per day, particularly in the rangefrom 0.1 to 5 mg/kg of body weight per day, will often be suitablealthough higher doses than this, for example in the range from 0.1 to 50mg/kg of body weight per day (or possibly even as high as described inU.S. Pat. No. 4,197,249) may be considered in view of the lower level oftoxic side effects obtained with the compounds. This dosage regime maybe continued for however many days is appropriate to the patient inquestion, the daily dosages being divided into several separateadministrations if desired. Thus, for example, in the case of conditionssuch as advanced breast cancer, non-Hodgkin's lymphoma and hepatoma,treatment for one day followed by a repeated dose after an interval,such as 21 days, may be appropriate whilst for the treatment of acutenon-lymphocytic leukaemia, treatment over 5 consecutive days may be moresuitable.

A fourth aspect of the invention provides a compound according to thefirst aspect of the invention for use in medicine (clinical and/orveterinary).

A fifth aspect of the invention provides a compound according to thefirst aspect of the invention for use as a cytotoxin, or a hypoxiaactivated prodrug thereof.

In one embodiment, the compound is for use in vivo as a cytotoxin, or ahypoxia activated prodrug thereof.

By “hypoxia activated prodrug thereof” we include that the compound ispreferentially cytotoxic under, or following exposure to, hypoxicconditions (i.e. exhibits greater cytotoxicity under, or followingexposure to, hypoxic conditions). For example, N-oxide compounds of theinvention, such as those of formulae V, VI, IX and X, are relativelynon-cytotoxic under normoxic conditions but are readily reduced underhypoxic conditions to generate cytotoxic compounds, such as those offormulae III, IV, VII and VIII.

In this context, “hypoxia” may be regarded as an oxygenation level of 4%or lower (or ≤23 mmHg) when measured directly by electrode methods. Forexample, the level of oxygenation may be lower than 3.0%, 2.5%, 2%,1.5%, 1% or 0.5 or 0.1%.

It will be appreciated by persons skilled in the art that thehypoxia-induced activation of a compound's cytotoxic activity may bedetermined either in vitro or in vivo.

For example, cytotoxicity may be determined in vitro at variousoxygenation levels measured by direct electrode methods.

Alternatively, the level of oxygenation in a tissue may be measuredindirectly, for example using histological sections probed with anenzyme detection assay or by gene expression analysis.

For confirmation of hypoxia-activated cytotoxicity in vivo, oxygenationlevels in living tissue may be determined using both the Helzel andOxyLite systems (for example, see Wen et al., 2008, Radiat. Res.169:67-75).

The results of blood flow and perfusion analyses may also infer theexistence of hypoxia in a given tissues. The application of agents thatmodify blood flow or compromise blood vessel formation would also onfirst principles be expected to reduce oxygenation in affected tissues.

In particular, the invention provides a compound according to the firstaspect of the invention for use in the treatment of cancer in mammals(most notably in humans).

For example, the compound may be for use in the treatment of a cancerselected from the group consisting of bladder cancer, breast cancer,bone cancer (primary and secondary, such as osteosarcoma and Ewingssarcoma), brain cancer (including glioblastoma multiforme andastrocytoma), cervical cancer, choriocarcinoma, colon and rectal cancer,endometrial cancer, eye cancer, gallbladder cancer, gastric cancer,gestational tumours, head and neck cancer, kidney (renal cell) cancer,laryngeal cancer, leukaemias (such as ALL, AML, CLL, CML and hairy cellleukaemias), liver cancer, lung cancer, lymphomas (such as Hodkin'slymphoma and non-Hodkin's lymphoma), melanoma, mesothelioma, mouthcancer, myeloma, nasal and sinus cancers, nasopharyngeal cancer,oesophageal cancer, ovarian cancer, pancreatic cancer, penile cancer,prostate cancer, stomach cancer, testicular cancer, thyroid cancer,uterine cancer, vaginal cancer, vulvar cancer and womb cancer.

In one embodiment, the compound is for use in the treatment of a solidtumour, such as various forms of sarcoma and carcinoma.

The compounds of the invention may be of particular use in the treatmentof a tumour that is naturally hypoxic, at least in part (for example,having a median oxygen level of below 3%, e.g. lower than 2.5%, 2%,1.5%, 1% or 0.5%). An example of such tumours are pancreatic cancer andprostate cancer, both typically exhibiting low oxygen levels and apropensity for malignant progression.

The hypoxia-activated cytotoxicity of the prodrug compounds of theinvention allows the cytotoxicity to be targeted to the tumour cells,reducing the risk of damage to healthy cells.

It is believed that hypoxia may play a role in facilitating themalignant progression of certain cancers (for example, see Rudolfsson &Bergh, 2009, Exp. Opin. Ther. Tar. 13:219-225). By exerting a cytotoxiceffect preferentially within the regions of tumour hypoxia, thecompounds of the invention may be able to target cancer cells that areotherwise resistant to treatment, e.g. by radiotherapy or conventionalchemotherapeutic agents. Eradication of such resistant cells may, inturn, lead to a reduction in metastasis.

Thus, in one embodiment, the compounds are for use in the treatment orprevention of metastases (which may arise from the aetiology of thecancer or as a consequence of treatment).

It will be appreciated by persons skilled in the art that the compoundsof the invention may be used on their own or in combination with othercancer treatments (such as radiotherapeutic modalities, e.g.radioisotopes and external beam radiation, and chemotherapeutic agents;see below).

In one embodiment, the compounds are for use as a monotherapy (i.e.without any other cancer treatments). However, it will be appreciatedthat the cancer patient may also be receiving different types ofbeneficial medication (such as a painkiller, sedative, antidepressant,antibiotic, etc).

However, the compounds of the invention may alternatively be for use incombination with one or more additional cancer treatments. For example,the compounds may be used in combination with one, two, three, four,five or more additional cancer treatments.

By “in combination” we include that the compound is administered to asubject who is receiving one or more additional cancer treatments in thesame course of therapy. Thus, the term covers not only the concomitantadministration of the compound with one or more additional cancertreatments (either as bolus doses or infusions) but also the temporallyseparate administration of these cancer treatments. For example, thecompound may be administered within a treatment schedule/cycle asdefined by the patient's oncologist to include one or more additionalcancer treatments, administered either before, concomitantly with orafter the compound; for example within ten weeks, nine weeks, eightweeks, seven weeks, six weeks, five weeks, four weeks, three weeks, twoweek, ten days, one week, five days, four days, three days, two days,one day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 10 minutes or fiveminutes. Each treatment cycle may be repeated on several occasions,normally up to 6 cycles, but could be more or less than this numberdepending on the nature of the cancer and its response to treatment.

It will be appreciated by persons skilled in the art that the one ormore additional cancer treatments may be chemotherapeutic agents orradiotherapeutic modalities.

In one embodiment, however, the one or more additional cancer treatmentscomprise or consist of one or more chemotherapeutic and/orradiotherapeutic modality.

Given the hypoxia-activated cytotoxicity of the prodrug compounds of theinvention, it is advantageous to administer them as part of acombination treatment with one or more chemotherapeutic agents and/orradiotherapeutic modalities capable of decreasing (at least,transiently) tumour oxygenation levels in vivo. For example, the one ormore chemotherapeutic agents and/or radiotherapeutic modalities may becapable of lowering the median oxygen level of the tumour to below 3%,for example below 2.5%, 2%, 1.5%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or below0.1%.

It will be appreciated by skilled persons that a reduction in tumouroxygenation levels may be achieved by a number of different means, forexample by the disruption of established tumour vasculature, preventionof angiogenesis (new blood vessel formation) and/or vasoconstriction.

Suitable cancer treatments may be selected from the group consisting ofanti-androgens (steroidal and non-steroidal), vascular disruptingagents, anti-angiogenic agents, anti-VEGFR agents, IL8 inhibitors, NOsynthase inhibitors, vasoconstricting agents, vasodilating agents andradiotherapy.

By “steroidal anti-androgens” we include cyproterone acetate.

By “anti-angiogenic agents” we include:

-   -   (a) anti-VEGF antibodies or antibody fragments such as        bevacizumab, axitinib, pazopanib and ranibizumab, pegaptanib        sodium, tryptophanyl-tRNA synthetase, AdPEDF, EYLEA, AG-013958,        JSM6427, TG100801, ATG3, rapamycin, endostatin;    -   (b) drugs that block signalling within the cell such as        lapatinib, sunitinib, sorafenib, axitinib, pazopanib and AZ2171;    -   (c) tetrahydrocannabinol (THC) and cannabidiol;    -   (d) thiazolidinediones such as rosiglitazone, pioglitazone and        troglitazone    -   (e) erlotinib, imatinib, gefitinib, dasatinib, nilotinib,        lapatinib; and    -   (f) drugs that affect signals between cells, such as thalidomide        and lenalidomide.

By “vascular disrupting agents” we include small molecules (such astaxanes, taxol, paclitaxel combretastatins, CA4P, Oxi4503, aurostatins,dolostatins, colchine, azacolchicinol, ZD6126I, MMP-activatedcolchicines, ICT2588, DMXAA, TZT1027 and AVE8062) and biologicals (suchas ADEPT, GDEPT and antibody drug-conjugates that target the tumourvasculature).

By “IL8 inhibitors” we include repertaxin.

By “NO synthase inhibitors” we include N^(G)-methyl-1-argininehydrochloride (546C88; 1-NMMA), NG-nitro-L-arginine (L-NNA),L-nitroarginine methyl ester (L-NAME), LG-nitro-L-arginine (L-NO-Arg)and 7-Nitro-Indazole (7-NI).

By “vasoconstricting agents” we include alpha 1 adrenoceptor agonists(e.g. methoxamine, phenylephrine, oxymetazoline, tetrahydralazine,xylometazoline), alpha 2 adrenoceptor agonists (e.g. clonidine,guanabenz, guanfacine, α-methyldopa) and vasopressin analogues (e.g.arginine vasopressin and triglycyl lysine vasopressin).

By “vasodilating (‘vascular steal’) agents” we includealpha-adrenoceptor antagonists (alpha-blockers), angiotensin convertingenzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs),beta2-adrenoceptor agonists (β2-agonists), calcium-channel blockers(CCBs), centrally acting sympatholytics, direct acting vasodilators,endothelin receptor antagonists, ganglionic blockers, nitrodilators,phosphodiesterase inhibitors, potassium-channel openers and renininhibitors.

By “radiotherapy modalities” we include conventional external beamradiation therapy (2DXRT), stereotactic radiosurgery (SRS), stereotacticbody radiation therapy (SBRT) and particle therapy such as protontherapy; brachytherapy such as SAVI™, MammoSite™, Contura™, Proxcelan™,TheraSeed™ and I-Seed™; radioisotope therapy such asmetaiodobenzylguanidine (MIBG), iodine-131, hormone-bound lutetium-177and yttrium-90 (peptide receptor radionuclide therapy).

In one preferred embodiment, the one or more cancer treatments is/arenon-steroidal anti-androgens, such as flutamide, nilutamide,bicalutamide, finasteride, dutasteride, bexlosteride, izonsteride,turosteride, epristeride and abiraterone.

Thus, in one embodiment, a compound according to the first aspect of theinvention is used in combination with bicalutamide in the treatment ofcancer, e.g. the prevention or reduction of metastasis.

Thus, in one embodiment, a compound according to the first aspect of theinvention is used in combination with cancer chemotherapeutic agentsand/or radiotherapeutic modalities and/or methods to reduce or increasethe air being breathed by the patients e.g. carbogen (with or withoutnicotinamide).

A related, sixth aspect of the invention provides the use of a compoundof the first aspect of the invention in the preparation of a medicamentfor treating cancer.

Preferred embodiments of the sixth aspect of the invention are describedabove in relation to the fifth aspect of the invention.

A seventh aspect of the invention provides a method of treating cancerin a patient comprising administering to the patient a therapeuticallyeffective amount of a compound of the first aspect of the invention.

In one embodiment, the patient is mammalian (e.g. human).

Preferred embodiments of the seventh aspect of the invention aredescribed above in relation to the fifth aspect of the invention.

An eighth aspect of the invention provides the use of a compound of thefirst aspect of the invention as a marker of the oxygenation level ofcells. In particular, such compounds may be used as a cellular hypoxicmarker, either in vitro or in vivo.

In one embodiment, the cells are mammalian (e.g. human).

Exposure of the N-oxide forms of the compounds of the invention (such asthose of formulae V and VI) to hypoxic cells causes their reduction tothe corresponding amine form (such as those of formulae III and IV),which can be readily detected by known means.

The presence of the reduced compound (such as those of formulae III andIV) can be used to detect hypoxic cells in vitro or in vivo. The innatefluorescence properties retained by the reduced compound(s) and theintracellular persistence of the reduced compound(s) are advantageousfor the discrimination, quantification and localisation of cells thathave been exposed to, or continue to be exposed to hypoxic conditions.

For example, when acting as a cellular marker for hypoxia, the reducedcompound (such as those of formulae III and IV) maybe detected usingmethod(s) that identify chemical composition or physical properties thatinclude but are not limited to mass spectrometry, infrared spectroscopy,colorimetry, Raman spectroscopy, nuclear magnetic resonance or positronemission tomography. Affinity capture methods would exploit the highaffinity binding potential of the reduced compound to DNA or syntheticpolynucleotide sequences.

Optical properties of the reduced compound(s) may be used to detectcompound in biological samples and include but are not limited to flowcytometry and microscopy utilising the innate fluorescent properties ofthe reduced compound. Secondary methods of detection of reduced compoundinclude but are not limited to a combination with other molecularreporter compounds with the reduced compound participating in resonantenergy transfer reactions as either an acceptor or donor. Othersecondary methods of detection of reduced compound include but are notlimited to methods using antibody based methods for molecular detection.

In one embodiment, the compounds of the invention are used to identifyhypoxic tumour cells in vivo, which may then be visualised in situ orexcised surgically.

In a further embodiment, a compound of the first aspect of the inventionis used as a cellular hypoxic marker in combination with anon-deuterated form of a compound of the first aspect of the invention.

By “in combination” in this context this includes that the compounds maybe applied to the cells (e.g. administered to a patient) eitherconcomitantly or sequentially (for example, within 24 hours, 12 hours, 6hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 30 minutes, 10minutes or less).

Thus, in a preferred embodiment, a compound of formulae IX or X is usedas a cellular hypoxic marker (in vivo or in vitro) in combination with acompound as disclosed in U.S. Pat. No. 5,132,327 (for example, AQ4N).

A related, ninth aspect of the invention provides a kit of parts for usein detecting the oxygenation level of cells comprising a compoundaccording to the first aspect of the invention.

Optionally, the kit further comprises a non-deuterated form of acompound according to the first aspect of the invention (such as acompound as disclosed in U.S. Pat. No. 5,132,327, for example AQ4N).

Preferably, the compound(s) is/are provided in a sterile, pyrogen-freeform.

It will be appreciated that the kits of the invention may furthercomprise one or more regents, control samples and/or instructions.

DESCRIPTION OF THE DRAWINGS

Preferred, non-limiting examples which embody certain aspects of theinvention will now be described, with reference to the followingfigures:

FIG. 1: The metabolites AQ4 and OCT1001 have similar cell cyclearresting actions, under normal oxygenation conditions, indicating thatselective deuteration has not modified intrinsic biological activity.

See Example B

FIG. 2: Similar hypoxia-enhanced cytotoxicity for AQ4N and OCT1002

See Example B

FIG. 3: Exemplification of that the bioactivity of AQ4N and OCT1002 isdependent upon the degree of hypoxia

See Example B

FIG. 4: Hypoxia-dependent growth inhibition by AQ4N and OCT1002 arisesfrom a similar mechanism of cell cycle arrest and is dependent on thedegree of hypoxia

See Example B

FIGS. 5 (A & B): Exemplification of shared bioactivity of AQ4N andOCT1002 under hypoxic conditions for functional p53 (DoHH2) and mutantp53 (SU-DHL-4) human B cell lymphoma cells

See Example B

FIG. 6: Intracellular accumulation of the OCT1001 far-red fluorescentchromophore under hypoxia is responsive to OCT1002 pro-drug dose andoxygenation level

See Example B

FIG. 7: Deuteration does not affect the intrinsic capacity of themetabolite (AQ4 or OCT1001) to accumulate within a cell

See Example B

FIG. 8: Accumulation of converted pro-drug OCT1001 correlates withgrowth arrest

See Example

FIGS. 9 (A & B): Demonstration of intracellular fluorescence followingexposure to OCT1002 under hypoxic conditions and that prodrugdeuteration reduces intracellular accumulation but increases persistenceof the metabolite.

See Example B

FIG. 10: Effect of bicalutamide on the oxygenation of 22Rv1 prostatetumours grown as xenografts

See Example C

FIG. 11: Effect of bicalutamide on blood vessels in 22Rv1 tumourxenografts

See Example C

FIG. 12: Effect of bicalutamide only or AQ4N single dose or OCT1002single dose on 22Rv1 xenografts in mice

See Example C

FIG. 13: Combined effect of AQ4N single dose or OCT1002 single dose on22Rv1 xenografts in mice treated daily with bicalutamide

See Example C

FIG. 14: Effect of OCT1002 on LNCaP xenografts in mice treatedwith/without bicalutamide

See Example C

FIG. 15: OCT1002 is reduced in hypoxic LNCaP tumour cells in vivo

See Example C

FIG. 16: OCT1002 reduces the metastatic spread of LNCaP tumours to thelungs

See Example C

DETAILED DESCRIPTION OF THE EMBODIMENTS Examples Example A: Synthesis ofAlkylaminoalkylaminoanthraquinones and their N-Oxides (a) Preparation of1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione

A mixture of 4,7-difluoroisobenzofuran-1,3-dione (8.50 g, 46.2 mmol),hydroquinone (5.64 g, 51.3 mmol), aluminium trichloride (36.9 g, 277mmol) and sulfolane (10 mL) was stirred together for 16 hours at 165° C.The reaction was effectively a melt as the mixture does not become aviscous red syrup until ˜150° C. To minimise the risk of a suddenexotherm and evolution of HCl gas, the reaction was stirred in portions,cooled in an ice bath and stirred again until mixing was sufficient.Only then was the mixture heated.

The mixture was poured carefully into ice and 2M HCl added (50 mL). Themixture was stirred, then filtered, washing the resultant slurry withfurther 2M HCl. The solid was re-slurried a further 3 times with 2M HClto reduce the aluminium content of the product. A final slurry waswashed with ether twice; drying in a round bottom flask at 60° C. untilconstant weight afforded 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione(9.82 g, 35.6 mmol, 77% yield).

¹H NMR (DMSO-d⁶) was clean and consistent with the desired material.

(b) Preparation of1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione

A suspension of deuterated-d6-dimethylamine hydrochloride (18.4 g, 210mmol) and 2-bromoacetonitrile (14.63 ml, 210 mmol) in anhydrous THF (250mL) in a round bottom flask was cooled to −10° C. with vigorous stirringand treated portion-wise with potassium carbonate (58.1 g, 420 mmol).After addition of the base, the reaction was fitted with a refluxcondenser and balloons and allowed to warm slowly to 5° C. over 2 hours.TLC (1:1 EtOAc/Iso-Hexanes) indicated the presence of product. Themixture was stirred at room temperature over a weekend.

The residue was diluted with DCM (250 mL) and filtered, washing withcopious amounts of DCM. The mother liquors were degassed with N₂ for 1hour, then reduced in volume by half on the rotavap. Then a 4M dioxanesolution of hydrogen chloride (52.5 ml, 210 mmol) was added,precipitating a white solid and the mixture allowed to stand for 10minutes before being filtered, washing with DCM to afforddeuterated-d6-dimethylacetonitrile (21.73 g, 172 mmol, 82% yield).

¹H NMR (400 MHz, d₆-DMSO) δ: 4.47 (2H, s) was consistent with thedesired material.

(c) Preparation of deuterated-d6-N,N-dimethylethylenediamine

To a stirred suspension of deuterated-d6-dimethylacetonitrile (21.72 g,172 mmol) in Et₂O (200 mL) at 0° C. was added d/w a 1M ether solution oflithium aluminium hydride (515 ml, 515 mmol) via dropping funnel over1.5 hours. After the addition, the cooling bath was removed. After afurther 1.5 hr, the reaction was quenched at 15° C. (no higher than 18°C.) with sodium sulfate decahydrate (0.5 eq rel. to LiAlH4, 80 g)cautiously (delayed reaction) over 1.5 hours. The mixture was left tostir for 1 hour and subsequently filtered, washing with ether. Thefiltrate was stored overnight in the dark. The ether was removed on therotavap at ˜40° C. with no vacuum to afforddeuterated-d6-N,N-dimethylethylenediamine (15.89 g, 160 mmol, 93% yieldwas clean and consistent with the desired material but contained ˜0.25eq ether).

¹H NMR (400 MHz, CDCl₃) δ: 2.76 (2H, t), 2.33 (2H, t)

(d) Preparation of1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione(“OCT1001”)

A solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, (4.9 g,17.74 mmol) in pyridine (35 mL) was treated withdeuterated-d6-N,N-dimethylethylenediamine, (16.57 ml, 142 mmol) as asteady stream. The mixture was warmed to 40° C. and allowed to stir for24 hours under a flow of nitrogen. The reaction was taken off heat andcooled in an ice-bath. A chilled mixture of ammonium hydroxide (30%, 30mL) and brine (30 mL) were added and the mixture stirred in an ice-bathfor 2 hours. After this time the mixture was filtered washing with a 10%ammonium hydroxide solution (130 mL). The solid was air-dried for 30minutes, then transferred to a tared flask and dried under vacuum at 60°C. until constant weight (˜2 h).

The bulk material was purified by flash chromatography (Biotage, 120 g)loading in DCM (through cotton wool plug) eluting with 6 then 10% MeOH(containing 1% NH₃)/DCM to give1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione(2.01 g, 4.73 mmol, 26.7% yield).

The product was analysed by LCMS (m/z 425.3 (M+H)⁺ (ES⁺); 423.2 (M−H)−(ES)−, at 0.90 and 1.03 min (product smears on column), 100%.

¹H NMR (CDCl₃) was clean and consistent with the desired material ¹H NMR(400 MHz, CDCl₃) δ: 13.51 (2H, s), 10.40 (2H, br t), 7.17 (2H, s), 7.11(2H, s), 3.47 (4H, q), 2.66 (4H, t).

(e) Preparation of1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione(“OCT1002”)

A suspension containing magnesium monoperoxyphthalate, MMPP (3.10 g,6.27 mmol) in methanol (8 mL) was added dropwise to a stirred solutionof 281-041 (1.90 g, 4.48 mmol), AQ4 in methanol (8 mL) and DCM (30 mL)cooled to −11° C. After the addition was complete, the reaction solutionwas allowed to warm to 0° C. and stirred overnight in the dark (warmedto room temperature during this time). Pre-cooled EtOAc (30 mL) and EtOH(6 mL) were added the reaction mixture at 0° C. This mixture was allowedto stir for 30 minutes then a 4M solution of hydrogen chloride (4.48 ml,17.90 mmol) in dioxane was added dropwise at approximately −10 to −15°C. The resulting slurry was then stirred for 10 minutes then filtered,washing with EtOH/Water (9:1, 100 mL), MeOH/EtOAc (1:1, 100 mL) andEtOAc (60 mL) and dried under vacuum (on rotavap) at 40° C. for 2 hours(constant weight) to afford1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]-amino)-5,8-di-hydroxyanthracene-9,10-dione(2.15 g, 3.99 mmol, 89% yield) as a dark blue powder.

The product was analysed by LCMS (standard 4 min. method, agilent), m/z458.2 (M+H)⁺ (ES⁺), at 3.07 min, 98.3% purity @ 254 nm. ¹H NMR (400 MHz,D₂O) δ: 6.73 (2H, br s), 6.43 (2H, br s), 3.76 (4H, br s), 3.58 (4H, brs).

¹H NMR (D₂O) was consistent with the desired material.

Example B: In Vitro Properties of1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dioneand Its Active Metabolite

-   -   (a) The metabolites AQ4 and OCT1001 have similar cell cycle        arresting actions, under normal oxygenation conditions,        indicating that selective deuteration has not modified intrinsic        biological activity.        -   A549 human lung cancer cells were cultured using            conventional methods for adherent cells and exposed for 4            days to 0, 1, 3 or 10 nM agents under standard cell culture            conditions of 5% carbon dioxide in air at 37 deg C.            Harvested cells were permeabilised and stained with the DNA            fluorescent dye ethidium bromide and cell cycle            distributions determined by conventional flow cytometry.        -   FIG. 1 (flow cytometry) shows similar increases in the G2            peaks of the DNA content distributions between 3-10 nM            (indicating cell cycle arrest) for cells exposed to            exogenous metabolites            1,4-bis-{[2-(dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione            (“AQ4”) and            1,4-bis-{[2-(deuterated-d6-dimethylamino)-ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione            (“OCT1001”).            (b) Similar Hypoxia-Enhanced Cytotoxicity for AQ4N and            OCT1002    -   Human T cell leukemia cells (Jurkat) were cultured using        conventional methods for suspension cultures in air or under 1%        oxygen conditions for 4 days in the presence of a range of        concentrations of either AQ4N or OCT1002. The relative cell        number was determined using a conventional Coulter Counter        particle counting method.    -   FIG. 2 shows that the compounds tested require hypoxic        conditions for the inhibition of cell proliferation. Thus,        1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione        (“AQ4N”) and        1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione        (“OCT1002”) both exhibit pronounced cytostatic activity under        conditions of hypoxia (1% oxygen).    -   As a control it is shown that hypoxia does not modify the        cytostatic action of a direct acting DNA topoisomerase inhibitor        (VP-16), achieving similar levels of prolonged cytostatic        action.        (c) Exemplification of that the Bioactivity of AQ4N and OCT1002        is Dependent Upon the Degree of Hypoxia    -   A549 human lung cancer cells were cultured using conventional        methods for adherent cells and exposed for 4 days to varying        concentrations of either AQ4N and OCT1002 agents under standard        cell culture conditions of 5% carbon dioxide in air (normoxia)        at 37 deg C., or under conditions of reduced oxygen (1% and 3%).    -   Data are plotted as relative population doublings determined by        cell detachment and Coulter Counter particle counting of cell        densities at the start and end of the exposure period.    -   FIG. 3 shows that for the compounds tested, namely        1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione        (“AQ4N”) and        1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione        (“OCT1002”), growth inhibition is dependent upon the degree of        hypoxia and drug concentration, with the two agents showing        similar responses.        (d) Hypoxic Sensitisation by AQ4N and OCT1002    -   A549 human lung cancer cells were used in this experiment;        culture conditions were as described in (c) above.    -   Cell cycle analysis was performed as described in (a) above.    -   FIG. 4 shows that the compounds tested, namely        1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione        (“AQ4N”) and        1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione        (“OCT1002”), generate similar cell cycle arrest (determined by        flow cytometry) within the bioactive drug dose range.    -   The degree of late cell cycle arrest is increased as oxygenation        levels are reduced.        (e) Exemplification of Shared Bioactivity of AQ4N and OCT1002        Under Hypoxic Conditions for p53 Functional and Mutant p53 Human        B Cell Lymphoma Cell Lines    -   Human B cell lymphoma cells were cultured using conventional        methods for suspension cultures in air, 1% or 3% oxygenation        conditions for 4 days in the presence of a range of        concentrations of either AQ4N or OCT1002. The relative cell        numbers were determined using a conventional Coulter Counter        particle counting method.    -   FIG. 5(A) shows that the compounds tested are equally and        selectively cytotoxic in hypoxic conditions against DoHH2 human        B cell lymphoma cells (bcl2 overexpressing; p53 wt) grown in        suspension and exposed to prodrugs for 4 days under 21%        (circles), 3% (triangles) or 1% O₂ (squares). Thus,        1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione        (“AQ4N”) and        1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione        (“OCT1002”) both exhibit pronounced cytostatic activity under        conditions of hypoxia (1% oxygen), with the growth inhibition        being sensitive to the degree of hypoxia.    -   Likewise, FIG. 5(B) shows that the prodrugs AQ4N and OCT1002 are        equally selectively cytotoxic in hypoxic conditions against        SU-DHL-4 human B cell lymphoma cells (bcl2 overexpressing; p53        mutant) grown in suspension and exposed to prodrugs for 4 days        under 21% (circles), 3% (triangles) or 1% O₂ (squares). Again,        the growth inhibition is sensitive to the degree of hypoxia.        (f) Reciprocity Between an Imposed pO₂ Level and the Degree of        End-Product Generation    -   OCT1002 and AQ4N show reciprocity between an imposed pO₂ level        and the degree of end-product generation in the biologically        relevant range of hypoxia with low or undetectable levels of        conversion under normoxia (and undetectable levels of AQ4N or        OCT1002 showing that the metabolites are the primary persistent        anthraquinone forms)    -   Relative to AQ4N, the deuterated variant OCT1002 shows a        reduction in overall capacity for reduction/accumulation (HPLC        analysis) within moribund cells, under protracted exposure        conditions showing a reduction of ‘redundant targeting’ in a        human lung cancer cell line. In this case redundant targeting of        a prodrug refers to the over-generation of the cytotoxic form        beyond that required for cell inactivation since conversion of        the prodrug can continue even when cell cycle arrest has        occurred. The consequences of over-generation will be increased        deleterious effects of the converted form when released from the        initial target cell. This undesirable bystander effect on nearby        tissue not initially subject to hypoxic conditions will comprise        non-target normal and tumour cells. Damage to normal cells is        clearly undesirable. Suboptimal exposure of non-target tumour        cells through a bystander effect may compromise their responses        to other agent(s) delivered in combination or generate selective        conditions for the development of drug resistance.    -   Table 1 shows a comparison of HPLC analysis of metabolite        generation following exposure of human A549 cells to AQ4N and        OCT1002 under varying degrees of hypoxia and concentration (data        derived from two determinations) where 21% is taken to represent        normal oxygenation conditions.    -   Data show the consistent reduction in the generation of OCT1001        compared with AQ4 in cells exposed to the conditions indicated        and washed prior to assay for the presence of prodrug or their        metabolites. Data also shows that the molecular forms present in        cells experiencing hypoxia are the metabolites and not parent        prodrugs.

TABLE 1 Dose of Humidified Relative prodrug oxygenation pmolesmetabolite generated prodrug (OCT1002 conditions per 10⁵ cells^(a)reduction to or AQ4N) pO₂ mm range range metabolite nM × days % O₂ HgAQ4 OCT1001 AQ4 OCT1001 OCT1001/AQ4 30 1% 7.1 9.25 5.64 1.46 1.20 0.6130 3% 21.4 0.78 0.49 0.05 0.06 0.62 30 21% 142.2 <0.10 0.10 0.03 0.021.02 100 1% 7.1 >42.95 16.17 6.59 8.16 <0.38 100 3% 21.4 5.58 1.93 1.130.16 0.35 100 21% 142.2 0.23 0.11 0.08 0.03 0.50 ^(a)No AQ4N or OCT1002detected in any sample indicating that either all prodrug forms aredepleted by undergoing metabolism or that, by the method used, suchforms are not readily retained within cells.(g) Intracellular Accumulation of the OCT1001 Far-Red FluorescentChromophore Under Hypoxia is Responsive to OCT1002 Prodrug Dose andOxygenation Level

-   -   Adherent A549 cells were cultured by conventional methods and        exposed to 0, 30 or 100 nM OCT1002 for 4 days in air, 1% or 3%        oxygenation levels. Detached cells were analysed far red        fluorescence intensity using conventional flow cytometry and 633        nm wavelength excitation (1×10⁴ cells analysed).    -   FIG. 6 shows mean fluorescence intensity increases in a linear        function of pro-drug dose and is dependent upon oxygenation        levels. This provides a convenient fluorometric, single live        cell analytical method for analyzing cell population experience        of prevailing pO₂ levels.        (h) Deuteration does not Affect the Intrinsic Capacity of the        Active Metabolite (OCT1001) to Accumulate    -   A549 human lung cancer cells were used in this experiment, as        described in (g) above.    -   Under normoxia conditions, similar levels of accumulation of        OCT1001 and AQ4 were observed within cells (see FIG. 7). Thus,        the overlaid histograms for the population distribution of        fluorescence in cells exposed to AQ4 or OCT1001 under normoxia        shows similar cellular accumulation potential.        (i) Accumulation of Converted Pro-Drug OCT1001 Correlates with        Growth Arrest (Increasingly Moribund Cells)    -   A549 human lung cancer cells were used in this experiment, as        described in (g) above, with the exception that light side        scatter (488 nm wavelength) was collected versus fluorescence        intensity (>695 nm wavelength).    -   FIG. 8 shows collected flow cytometry data for A549 cells        exposed to 0, 30 and 100 nM OCT1002 under 21%, 3% and 1% oxygen        over 4 days.    -   Plotting all data points reveals that increasing light side        scatter parameter (reflecting the expansion of cell size and        complexity associated with growth arrest) correlates with the        increase in fluorescence intensity (indicating co-accumulation        of OCT1001).        (j) Demonstration of Intracellular Fluorescence Following        Exposure to OCT1002 Under Hypoxic Conditions and that Prodrug        Deuteration Reduces Intracellular Accumulation but Increases        Persistence of the Metabolite.    -   A549 cells were cultured using conventional methods and allowed        to attach to the glass substrate in chamber slides and exposed        to OCT1002 under hypoxia. Fluorescence imaging of live cells        used conventional confocal fluorescence microscopy using        red-line laser excitation.    -   FIG. 9a shows that the far red fluorescence detected in cells is        intracellular (background fluorescence not detectable in control        cultures) with evidence of regions of cytoplasmic accumulation.        The data exemplify the single cell hypoxia sensing properties of        the deuterated pro-drug at the single-cell level.    -   Given the confirmation of intracellular fluorescence associated        with conversion of OCT1002 to OCT1001 under hypoxia, A549 human        lung cancer cells were further used to assess differential        accumulation or retention of the metabolites using flow        cytometry as described in (g) above. Following exposure to AQ4N        or OCT1002 under 1% oxygen, cells were detached for analysis, or        washed and incubated for 24 h in drug free medium and held under        normal oxygenation conditions prior to detachment and analysis        by flow cytometry    -   Flow cytometry data in FIG. 9b shows the reduced cellular        accumulation (after 4 day exposure) but also reduced loss (after        24 h post exposure recovery) of intracellular fluorescence        attributable to the metabolite OCT1001, compared with the        fluorescence attributable to the metabolite AQ4, following        exposure of A549 cells to pro-drugs OCT1001 and AQ4 in A549        under hypoxia. Thus, deuteration changes the in situ        intracellular compartment loading/retention of hypoxia converted        forms of OCT1002.        Conclusions

The above studies demonstrate the in vitro properties of an exemplarydeuterated compound of the invention (the N-oxide prodrug, OCT1002, andits active metabolite, OCT1001).

-   -   (a) Evidence of primary biological activity following reduction        of the prodrug in hypoxia that elicits growth arrest in        different tumour cell types;    -   (b) For an equally effective toxicity for the reduced drug        (OCT1001) the toxicity of OCT1002 to cells in normoxia is        significantly less.    -   (c) Reciprocity between pO₂ level and end-product generation in        the biologically relevant range of hypoxia;    -   (d) The ability of cellular fluorescence to report in situ        generation of of metabolite providing for the sensing and        reporting of hypoxic environments;    -   (e) A distinct molecular/atomic signature provided by        site-specific deuteration that can be used to trace prodrug        conversion and metabolism by physico-chemical methods; and    -   (f) Prodrug deuteration results in reduced accumulation of the        reduced form under hypoxia but increased persistence/retention        of the reduced form upon removal of external drug and        re-oxygenation. This property demonstrated in moribund cells        confirms both reduced redundant targeting of the deuterated form        and convenient signal persistence for hypoxia sensing        applications.

Example C—Effect of OCT1002 on Tumour Growth and Metastasis In Vivo

Given the hypoxia-activated cytotoxicity of the prodrug compounds of theinvention, it may be advantageous to administer them as part of acombination treatment with one or more chemotherapeutic agents and/orradiotherapeutic modalities capable of decreasing (at least,transiently) tumour oxygenation levels in vivo. Bicalutamide (marketedas Casodex, Cosudex, Calutide, Kalumid) is an oral non-steroidalanti-androgen used in the treatment of prostate cancer including asmonotherapy for the treatment of earlier stages of the disease. 22Rv1 isa human prostate carcinoma epithelial cell line (Sramkoski R M, PretlowT G 2nd, Giaconia J M, Pretlow T P, Schwartz S, Sy M S, Marengo S R,Rhim J S, Zhang D, Jacobberger J W A new human prostate carcinoma cellline, 22Rv1. In Vitro Cell Dev Biol Anim. 1999 July-August;35(7):403-9). The cell line expresses prostate specific antigen (PSA).Growth is weakly stimulated by dihydroxytestosterone and lysates areimmunoreactive with androgen receptor antibody by Western blot analysis.

(i) Effect of Bicalutamide on the Oxygenation of 22Rv1 Prostate TumoursGrown as Xenografts

-   -   Male SCID mice (>8 weeks) bearing 22Rv1 prostate tumours of        100-150 mm³ were treated daily for 28 days by oral gavage with        either vehicle (0.1% DMSO in corn oil) or bicalutamide (2        mg/kg/day in vehicle).    -   Before commencement of treatment (day 0) pO2 (mmHg) was measured        using an Oxylite oxygen electrode probe; this was repeated on        the days indicated.

Day of Mean p0₂ ± SD Significance Significance Treatment Treatment(mmHg) (to vehicle) (to day 0) Vehicle only 0 15.277 ± 11.254 7 14.741 ±4.290  14 3.165 ± 3.275 21 2.660 ± 1.889 28 3.546 ± 1.563 Bicalutamide 015.277 ± 11.254 ns (2 mg/kg/ 7 1.996 ± 1.989 <0.05 <0.05 day) 14 0.486 ±0.107 ns <0.05 21 1.291 ± 0.291 ns <0.05 28 11.905 ± 0.861  <0.01 nsTable 3 shows mean pO2 values±SD. Also shown are statistical comparisonsof the bicalutamide group compared to control and to day 0 values;ns=not significant.

-   -   22Rv1 cells grow as a solid tumour on the backs of SCID mice.    -   Tumour oxygenation was measured over 28 days in vehicle and        bicalutamide (2 mg/kg/day) treated mice (see Table 3 above).    -   Bicalutamide caused a drop in tumour oxygenation (as shown in        FIG. 10); from ˜15.3 mmHg (2% oxygen) to 2.0 mmHg (0.3% oxygen)        at day 7 and to 0.5 mmHg (0.1% oxygen) at day 14. This drop        persists for approximately 2 weeks before recovering to almost        normal somewhere beyond 21 and 28 (at which time it is not        significantly different from the starting level of oxygenation).    -   The faster-growing, vehicle-treated, controls showed no        significant drop in oxygen levels up to day 7. However, during        the subsequent week (probably related to tumour size) the median        oxygen levels drop to about 3 mmHg (0.4% oxygen) and do indicate        recovery.        Conclusion        Hypoxia exists in the 22Rv1 solid tumour model. The addition of        bicalutamide alters the patterns of oxygen levels indicated by        the tumour. Hypoxia is clearly relevant to the 22Rv1 model and        the response of such a model to monotherapy (±bicalutamide); and        the potential role of OCT1002 in a combination treatment.        (ii) Effect of Bicalutamide on Blood Vessels in 22Rv1 Tumour        Xenografts    -   Dorsal skin folds were secured using window chambers onto the        backs of male SCID mice (>8 weeks). 22Rv1 tumour fragments were        implanted and allowed to vascularise for 7 days before        commencement of treatment.    -   Animals were treated daily via oral gavage with either vehicle        (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg in vehicle).    -   Anaesthetised mice were injected i.v. with FITC-labelled dextran        immediately prior to imaging with a confocal microscope.    -   Each image is representative of a minimum of 5 animals per        treatment group.    -   22Rv1 tumours were grown in window chambers/dorsal skin flaps on        the backs of SCID mice. Tumour fragments were imaged (see        FIG. 11) before treatment began (A) vehicle and (E) bicalutamide        pre-treatment groups and then after 7, 14 and 21 days of        treatment, (B-D) vehicle only (F-H) bicalutamide (10×        magnification).    -   Within 7 days tumour fragments showed the development of        extensive small vessels indicated as day 0 of the experimental        period (see FIG. 11).    -   In vehicle-treated tumours vessel density showed a slight change        by day 14 and by day 21 the small vessel numbers were reduced.    -   In bicalutamide-treated tumours, loss of small vessels was seen        at days 7 and 14 with some recovery by day 21. This is        consistent with oxygen electrode data i.e., fall and then        recovery of oxygenation.        Conclusions    -   Vehicle has no effect on blood vessels for at least 7 days. By        day 14 there is a slight pruning of vessels which is clearly        seen by day 21. This vessel loss, although not as dramatic as        seen in the bicalutamide treated tumours (at days 7 and 14; Ming        et al., 2007), may be due to vascular collapse and necrosis seen        at this time in this fast growing vehicle-treated tumour. The        oxygen levels drop somewhat earlier, i.e. sometime between days        7 and 14 (see FIG. 10).    -   In bicalutamide-treated 22Rv1 tumours there is a marked early        loss of tumour vasculature (by day 7). The data provide evidence        that bicalutamide causes a profound drop in tumour oxygenation        through an anti-vascular effect; this may be direct or        alternatively it could be due to inhibition of the production of        pro-angiogenic factors by the tumour cells.    -   By day 21, the small vessels have returned which is consistent        with the increased level of oxygenation seen in FIG. 10.        (iii) Effect of Bicalutamide Only or AQ4N Single Dose or OCT1002        Single Dose on 22Rv1 Xenografts in Mice.    -   Male SCID mice (>8 weeks) bearing 22Rv1 xenograft tumours of        100-150 mm3 were treated for 28 days. Treatment included Vehicle        (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day in vehicle)        both administered daily via oral gavage. Alternatively, at day 7        of the experimental period AQ4N or OCT1002 (50 mg/kg in sterile        PBS) was administered intraperitoneally as a single dose.    -   Tumour volumes were measured using callipers every other day.    -   Data analysis to determine the time dependent effect of        treatment(s) on tumour volume was performed. Tumour volume was        normalised to day 6 (ie pre-produg addition). Time series and        regression analysis was undertaken.    -   Tumour growth is normalised to day 6, so that overall tumour        growth, and patterns can be compared FIGS. 12 (A and B).    -   Despite the lack of sensitivity to bicalutamide in vitro, the        22Rv1 tumours show a small but significant slowing of growth.        Classical cross-sectional comparison of growth delay showed that        mice treated with vehicle required 14.0±0.3 days to reach four        times the volume at the start of treatment. Bicalutamide        treatment (2 mg/kg/day) increased this to 18.5±0.8 days; thus        this was a growth delay of 4.5 days.    -   Graphical regression fits indicate that 22RV1 tumours treated        with bicalutamide only show a delay in growth (during days        10-20), despite continuing daily exposure to bicalutamide; the        tumours exhibit an overall exponential growth pattern        (R²=0.9915) to day 24.    -   Addition of AQ4N given as a single dose (50 mg/kg) on day 7, a        different growth pattern was observed compared to that of the        bicalutamide treatment alone, regression fitting showed a        non-linear polynomial growth pattern (R²=0.9948).    -   Addition of OCT1002 given as a single dose (50 mg/kg) on day 7;        tumours treated with this single dose were capable of        maintaining a polynomial (x²) growth rate pattern, this was also        a non-linear pattern (R²=0.9978).    -   OCT1002 treated tumours showed an overall reduced rate of growth        over the remaining period of the experiment (beyond day 22)        compared to the bicalutamide only and AQ4N only treated tumours.        Cumulative growth over the entire period (progressive area under        the curve), indicates this difference (FIG. 12B).        (iv) Combined Effect of AQ4N Single Dose or OCT1002 Single Dose        on 22Rv1 Xenografts in Mice Treated Daily with Bicalutamide    -   Male SCID mice (>8 weeks) bearing 22Rv1 xenograft tumours of        100-150 mm³ were treated for 28 days. Vehicle (0.1% DMSO in corn        oil) and bicalutamide (2 mg/kg/day in vehicle) treatments were        administered daily via oral gavage.    -   AQ4N or OCT1002 (50 mg/kg in sterile PBS) was administered        intraperitoneally as a single dose at day 7.    -   Tumour volumes were measured using callipers every other day.    -   Animals were culled once the tumour burden reached ≥800 mm³.    -   Tumour growth is normalised to day 6, so that overall tumour        growth, and patterns can be compared (FIGS. 13 (A and B).    -   Bicalutamide treatment alone (2 mg/kg/day) is discussed above;        it exhibits a overall exponential growth pattern (R²=0.9915) to        day 24.    -   Bicalutamide treatment was combined with an AQ4N single dose (50        mg/kg) given on day 7, a modified growth pattern was observed        compared to that of the bicalutamide treatment alone, regression        fitting showed a non-linear polynomial growth pattern        (R²=0.9982), with divergence of growth to bicalutamide alone        apparent at beyond day 20.    -   Bicalutamide treatment treatment was combined with an OCT1002        single dose (50 mg/kg) given on day 7; a different modified        growth pattern was observed regression fitting showed a linear        tumour growth response (R²=0.9955), with divergence of growth to        bicalutamide alone apparent at beyond day 14.        Conclusions    -   The combined treatment indicates two critical features.

(i) the first is an earlier effective tumour growth inhibition ofOCT1002 on the bicalutamide treated tumours compared to AQ4N;

(ii) the second indicates a sustained tumour growth inhibition(indicated by a maintained linear response); that reflects a persistenceOCT1002 and tumour growth inhibition.

-   -   Thus with OCT1002 administered at the time when hypoxia/low        oxygen levels were achieved; an early and sustained effect was        obtained. The combination of OCT1002 with bicalutamide was more        effective at inhibiting tumour growth as compared to AQ4N with        bicalutamide.        (v) Effect of OCT1002 on LNCaP Xenografts in Mice Treated        with/without Bicalutamide    -   Male SCID mice (>8 weeks) bearing LNCaP xenograft tumours of        100-150 mm³ were treated for 28 days.    -   Vehicle (0.1% DMSO in corn oil) and bicalutamide (2 mg/kg/day in        vehicle) treatments were administered daily via oral gavage.        OCT1002 (50 mg/kg in sterile PBS) was administered        intraperitoneally as a single dose at day 7.    -   Tumour volumes were measured using callipers every other day.    -   Growth curves are the mean of ≥5 animals in bicalutamide and        vehicle treatment groups; bicalutamide+OCT1002 group (n=5 until        day 14; then n=3) and vehicle+OCT1002 (n=5 until day 13;        n=1)±s.e.    -   Table 6 below shows the growth delays calculated for the time to        reach twice the treatment size.    -   Bicalutamide causes a 5.1 day delay in LNCaP tumour growth        compared to vehicle.    -   When OCT1002 (50 mg/kg single dose on day 7) was given in        combination with vehicle (daily administration) there was no        appreciable effect on tumour growth (Table 6 below).    -   Bicalutamide (daily for 28 days) initially slows tumour growth        until day 12-14. Tumour growth then recovers and the tumours are        the same size as the vehicle-treated tumours by day 28 (Table 6        below).    -   Tumours treated with a single dose of OCT1002 reduced the growth        rate in combination with bicalutamide and this was significantly        different from control at all times between days 14 and days 28        at the termination of the experiment (FIG. 15).        Conclusions    -   Administration of OCT1002 at day 7 had no significant effect on        LNCaP tumour growth. This shows that the better-oxygenated        tumours (i.e. as compared to bicalutamide-treated tumours) there        is low toxicity of OCT1002 and that this better-oxygenated        fraction of cells is predominant in contributing to growth in        vehicle-treated control tumours.    -   Combination of a single dose of OCT1002 with bicalutamide        blocked the increase in growth rate observed in the        bicalutamide-treated group. OCT1002 is very effective at        blocking tumour growth from 12 days onwards where, for        bicalutamide alone, there is a delay and then recovery.    -   The initial slowing and then recovery after day 14 of LNCaP        tumour growth, during daily treatment with bicalutamide, is        consistent with the drop and then recovery of tumour oxygenation        and blood vessels (Ming et al., 2012, supra.).

TABLE 6 Time to 2x start volume Growth Treatment (days) Delay (days)Vehicle Only 11.2 ± 1.88 Bicalutamide 16.2 ± 1.94   5 ± 3.82 OCT1002only   13 ± 0.89 1.8 ± 2.77 OCT1002 + 25.5 ± 3.22 14.3 ± 5.1 Bicalutamide(vi) OCT1002 Prodrug is Converted to Metabolites in Hypoxic LNCaP TumourCells In VivoMethods

-   -   A dorsal skin flap (window chamber) was attached to the dorsum        of male SCID mice and a 1 mm³ LNCaP-Luc tumour fragment        inserted; this was left to vascularise for 7 days.    -   Mice were then treated orally for 21 days with either vehicle        (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day).    -   Seven days after induction of (a) vehicle or (b) bicalutamide        mice were dosed intraperitoneally with OCT1002 (50 mg/kg).    -   Two hours after injection of OCT1002 mice were injected        intravenously with FITC-dextran.    -   Images were captured using a confocal laser scanning microscopy        to show blood vessels (green) and OCT1001 (blue) patterns in the        tumour. (Magnification 10× with 3× digital zoom) (pixel        resolution).    -   Images were also acquired at day 0 (i.e. 7 days after tumour        fragment implantation), 14 and 21.    -   Only FITC-dextran was administered on days 0, 14 and 21. (c)        Full panel of images 0, 7, 14 and 21 days.    -   Control mice were treated orally for 21 days with vehicle (0.1%        DMSO in corn oil): vascularisation was maintained throughout. By        7 days the tumour fragment was vascularised (day 0 of experiment        shown in FIG. 15C green).    -   In mice treated with vehicle+OCT1002 at day 7: the converted        compound OCT1001 (blue) is in a few areas where vascularisation        is poor (FIG. 15A).    -   Mice treated with bicalutamide (2 mg/kg/day in vehicle):        vascularisation was reduced at days 7. On day 7, two hours after        intraperitoneal injection of a single dose of OCT1002 (50 mg/kg)        large quantities of converted compound (OCT1001; blue) can be        seen across the whole tumour fragment (FIG. 15B).    -   Mice treated with bicalutamide (2 mg/kg/day in vehicle):        vascularisation was reduced at days 7 and 14, this recovered by        day 21 (Ming et al., 2012, supra.).    -   Tumours were re-examined at days 14 and 21.    -   OCT1001 (blue) is still localised to the tumour at day 14; by        day 21 the amount of compound was considerably lower (FIG. 15C).        Conclusions    -   OCT1002, administered intraperitoneally, distributed widely        throughout the tumour fragments localised in the skin fold on        the backs of the mice.    -   Distribution was extensive even when the vasculature was        significantly decreased (i.e. by the bicalutamide treatment at        days 7 and 14).    -   OCT1001 was found predominantly where the oxygen levels are low        (i.e. areas of poor vascularisation); small areas were seen in        the control also (indicating that hypoxia can occur in untreated        tumours but to a lesser extent.    -   Extensive localisation of OCT1001 was still observed at day 14        of bicalutamide treatment showing that the compound remains for        at least 7 days.    -   By day 21, tumour blood vessels show some recovery and OCT1001        levels are lower although still above background.    -   The persistence of the reduced product, OCT1001, for >7 days        shows that the half-life of the converted compound is long.    -   However it may be less than AQ4 since by day 21 the OCT1001        signal is very much decreased.    -   This may be due to the different cellular binding properties of        OCT1001 as compared to AQ4 and potentially will provide a        rationale for less cumulative systemic toxicity which might be        caused through persistence of small amount of reduced compound        in marginally hypoxic peripheral tissues. This should not affect        the primary efficacy of OCT1002/OCT1001 at the predominant site        of metabolism (i.e. the hypoxic cells in tumours) since large        amounts are seen throughout the hypoxic tumour fragment which        persists for greater than 7 days.        (vii) OCT1002 Reduces the Metastatic Spread of LNCaP Tumours to        the Lungs        Methods    -   Male SCID mice (>8 weeks) bearing LNCaP-luc xenograft tumours of        100-150 mm³ were treated for 28 days (the luciferase-expressing        cells had similar characteristics to parental LNCaP cells; Ming        et al., 2012, supra.).    -   Vehicle (0.1% DMSO in corn oil) and bicalutamide (2 mg/kg/day in        vehicle) treatments were administered daily via oral gavage.    -   OCT1002 (50 mg/kg in sterile PBS) was administered        intraperitoneally as a single dose at day 7.    -   On day 28 of treatment, animals were injected i.p. with a        solution of D-luciferin (150 mg/kg in PBS) 15 mins prior to        imaging.    -   Animals were then killed and a range of tissues were removed for        the detection of bioluminescence using the IVIS imaging system        (Xenogen, USA).    -   Images were taken for 5 minutes and quantification of        bioluminescence was achieved by drawing a region of interest        around the area and measuring total flux in photons/second        (ph/sec).    -   A range of tissues were excised, however only the lungs and        tumour showed measurable bioluminescence. The mean±s.e of        bioluminescence in the lungs is shown in FIG. 16; bicalutamide        and vehicle treatment groups (n=10); bicalutamide+OCT1002 group        (n=3). and vehicle+OCT1002 (n=1). *Bicalutamide vs        bicalutamide+OCT1002 (p=0.024). Mice treated with vehicle showed        some metastatic spread to the lung. OCT1002, single dose day 7,        had no effect on this spread.    -   Bicalutamide appeared to increase the extent of metastatic        spread although the result did not reach significance.    -   Combination of OCT1002 with bicalutamide showed that OCT1002        significantly reduces the metastatic spread to the lungs caused        by bicalutamide. (P=0.024)        Conclusions    -   OCT1002 given as a single dose at day 7 was able to reduce        significantly the metastatic spread to the lungs caused by        bicalutamide treatment.

The invention claimed is:
 1. A compound having one of Formula III or V:

wherein each Y is independently selected from the group consisting ofhydrogen, hydroxy, halogeno, amino, C₁₋₄ alkoxy and C₂₋₈ alkanoyloxy. 2.The compound according to claim 1, wherein the compound is of FormulaVII or IX:


3. The compound according to claim 1 wherein each Y is independentlyselected from the group consisting of hydrogen, hydroxy and halogeno. 4.The pharmaceutical composition comprising the compound according toclaim 1 together with a pharmaceutically acceptable buffer, diluent,carrier, adjuvant or excipient.
 5. A kit for detecting the oxygenationlevel of cells comprising the compound according to claim
 1. 6. The kitaccording to claim 5 further comprising a non-deuterated form of acompound of Formula V.
 7. A method of treating a pancreatic cancertumour in a patient comprising administering to the patient atherapeutically effective amount of the compound according to claim 1.8. The method according to claim 7, wherein administering thetherapeutically effective amount treats metastases or reduces metastaticspread.
 9. The method according to claim 7 further comprisingadministering to the patient one or more of a chemotherapeutic agent anda radiotherapeutic agent in combination with the therapeuticallyeffective amount of the compound.
 10. The method according to claim 9wherein the one or more of the chemotherapeutic agent and theradiotherapeutic agent is/are selected from the group consisting ofanti-androgens (steroidal and non-steroidal), vascular disruptingagents, anti-angiogenic agents, anti-VEGFR agents, IL8 inhibitors, NOsynthase inhibitors, vasoconstricting agents, vasodilating agents, andradiotherapeutic modalities.
 11. The method according to claim 10wherein the one or more of the chemotherapeutic agent and theradiotherapeutic agent is at least one anti-androgen.
 12. The methodaccording to claim 11 wherein the at least one anti-androgen is selectedfrom the group consisting of flutamide, nilutamide, bicalutamide,finasteride, dutasteride, bexlosteride, izonsteride, turosteride,epristeride, abiraterone and combinations thereof.
 13. The methodaccording to claim 12 wherein the at least one anti-androgen isbicalutamide.
 14. The method according to claim 9 wherein the one ormore of the chemotherapeutic agent and the radiotherapeutic agentdecreases tumour oxygenation in vivo.
 15. The method according to claim14 wherein the one or more of the chemotherapeutic agent and theradiotherapeutic agent lowers the median oxygen level of the tumour tobelow 3%.
 16. A method of treating a prostate cancer tumour in a patientcomprising administering to the patient a therapeutically effectiveamount of the compound according to claim
 1. 17. The method according toclaim 16, wherein administering the therapeutically effective amounttreats metastases or reduces metastatic spread.
 18. The method accordingto claim 16 further comprising administering to the patient one or moreof a chemotherapeutic agent and a radiotherapeutic agent in combinationwith the therapeutically effective amount of the compound.
 19. Themethod according to claim 18 wherein the one or more of thechemotherapeutic agent and the radiotherapeutic agent is/are selectedfrom the group consisting of anti-androgens (steroidal andnon-steroidal), vascular disrupting agents, anti-angiogenic agents,anti-VEGFR agents, IL8 inhibitors, NO synthase inhibitors,vasoconstricting agents, vasodilating agents, and radiotherapeuticmodalities.
 20. The method according to claim 19 wherein the one or moreof the chemotherapeutic agent and the radiotherapeutic agent is at leastone anti-androgen.
 21. The method according to claim 20 wherein the atleast one anti-androgen is selected from the group consisting offlutamide, nilutamide, bicalutamide, finasteride, dutasteride,bexlosteride, izonsteride, turosteride, epristeride, abiraterone andcombinations thereof.
 22. The method according to claim 21 wherein theat least one anti-androgen is bicalutamide.
 23. The method according toclaim 18 wherein the one or more of the chemotherapeutic agent and theradiotherapeutic agent decreases tumour oxygenation in vivo.
 24. Themethod according to claim 23 wherein the one or more of thechemotherapeutic agent and the radiotherapeutic agent lowers the medianoxygen level of the tumour to below 3%.
 25. A process for making thecompound according to claim 1 comprising reacting ananthracene-9,10-dione with a deuterated alkylenediamine under conditionssuitable for the production of an alkylaminoalkyl-aminoanthraquinone.26. The process according to claim 25 further comprising the step ofreacting the alkylaminoalkylaminoanthraquinone with amonoperoxyphthalate under conditions suitable for the production of anN-oxide derivative of the alkylaminoalkylaminoanthraquinone.
 27. Theprocess according to claim 25 comprising reacting1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, with deuteratedN,N-dimethylethylenediamine under conditions suitable for the productionof1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione.28. The process according to claim 27 further comprising the step ofreacting the1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dionewith magnesium monoperoxyphthalate under conditions suitable for theproduction of1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione.29. A method of detecting hypoxic cells in vitro or in vivo in a groupof cells, the method comprising: exposing a compound of Formula V:

wherein each Y is independently selected from the group consisting ofhydrogen, hydroxy, halogeno, amino, C₁₋₄ alkoxy and C₂₋₈ alkanoyloxy, tothe group of cells; analyzing the cells for the presence of acorresponding reduced compound of Formula III:

determining the hypoxic cells based on the presence of the correspondingreduced compound.
 30. The method according to claim 29 in vitro.
 31. Themethod according to claim 29 in vivo.
 32. The method according to claim31, further comprising: surgically excising cells identified as beinghypoxic.
 33. The method according to claim 29 wherein the compound isused in combination with a non-deuterated form of a compound of FormulaV.
 34. The method according to claim 29 wherein the compound is detectedusing a method selected from the group consisting of mass spectrometry,nuclear magnetic resonance, infrared spectroscopy, colorimetrically,Raman spectroscopy, nuclear magnetic resonance, affinity capturemethods, immunohistochemistry, flow cytometry, microscopy andantibody-based detection methods.